Bottom Line:
Even better accuracies between 0.28 g/l - 0.59 g/l were found for independent test samples of an arbitrarily selected cultivation.ATR-FTIR was found to be suitable for the rapid analysis of rhamnolipids in a biotechnological process with good reproducibility in sample determination and sufficient accuracy.An improvement in accuracy through continuous expansion and validation of the reference spectra set seems very likely.

Affiliation: Research University Karlsruhe, Institute of Engineering in Life Sciences, Section of Technical Biology, Engler-Bunte-Ring 1, 76131 Karlsruhe, Germany. Rudolf.Hausmann@tebi.uni-karlsruhe.de.

ABSTRACT

Background: Vibrational spectroscopic techniques are becoming increasingly important and popular because they have the potential to provide rapid and convenient solutions to routine analytical problems. Using these techniques, a variety of substances can be characterized, identified and also quantified rapidly.

Results: The rapid ATR-FTIR (Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy) in time technique has been applied, which is suitable to quantify the concentrations of microbial rhamnolipids in a typical cultivation process. While the usually applied HPLC analysis requires an extensive and time consuming multi step extraction protocol for sample preparation, the ATR-FTIR-method allows the quantification of the rhamnolipids within 20 minutes. Accuracies between 0.5 g/l - 2.1 g/l for the different analytes were determined by cross validation of the calibration set. Even better accuracies between 0.28 g/l - 0.59 g/l were found for independent test samples of an arbitrarily selected cultivation.

Conclusion: ATR-FTIR was found to be suitable for the rapid analysis of rhamnolipids in a biotechnological process with good reproducibility in sample determination and sufficient accuracy. An improvement in accuracy through continuous expansion and validation of the reference spectra set seems very likely.

Figure 1: Rhamnolipid 3 IR-spectrum. ATR-FTIR spectrum of an aqueous solution (89 mg/mL) of rhamnolipid 3. The allocation of conspicuous absorption to corresponding characteristic group absorptions from literature was attempted.

Mentions:
An ATR-FTIR spectrum of pure rhamnolipid 3 in water was recorded, as shown in Figure 1. For a better understanding of the IR spectrum, subsequently the dominant absorbance bands were correlated to the according group absorbance frequencies. The broad negative bands at about 3300 cm-1 and 1640 cm-1 result from a higher water concentration in the reference spectrum (pure water) relative to the aqueous sample, and are attributed to hydrogen bonding and O-H stretching of water. The double bands at 2921 and 2855 cm-1 are derived from symmetric C-H stretching vibrations of aliphatic groups, like those represented in the hydroxydecanoic acid chain tails of rhamnolipid 3. A C=O stretching band at 1730 cm-1 is characteristic of ester bonds and carboxylic acid groups. In the fingerprint region of the spectrum, the area between 1200 – 1460 cm-1 represents C-H and O-H deformation vibrations, typical for carbohydrates as in the rhamnose units of the molecule, for example. The lower range of the fingerprint region below 1200 cm-1 represents different kinds of C-H, C-O and CH3 vibrations which cannot be allocated more closely [6].

Figure 1: Rhamnolipid 3 IR-spectrum. ATR-FTIR spectrum of an aqueous solution (89 mg/mL) of rhamnolipid 3. The allocation of conspicuous absorption to corresponding characteristic group absorptions from literature was attempted.

Mentions:
An ATR-FTIR spectrum of pure rhamnolipid 3 in water was recorded, as shown in Figure 1. For a better understanding of the IR spectrum, subsequently the dominant absorbance bands were correlated to the according group absorbance frequencies. The broad negative bands at about 3300 cm-1 and 1640 cm-1 result from a higher water concentration in the reference spectrum (pure water) relative to the aqueous sample, and are attributed to hydrogen bonding and O-H stretching of water. The double bands at 2921 and 2855 cm-1 are derived from symmetric C-H stretching vibrations of aliphatic groups, like those represented in the hydroxydecanoic acid chain tails of rhamnolipid 3. A C=O stretching band at 1730 cm-1 is characteristic of ester bonds and carboxylic acid groups. In the fingerprint region of the spectrum, the area between 1200 – 1460 cm-1 represents C-H and O-H deformation vibrations, typical for carbohydrates as in the rhamnose units of the molecule, for example. The lower range of the fingerprint region below 1200 cm-1 represents different kinds of C-H, C-O and CH3 vibrations which cannot be allocated more closely [6].

Bottom Line:
Even better accuracies between 0.28 g/l - 0.59 g/l were found for independent test samples of an arbitrarily selected cultivation.ATR-FTIR was found to be suitable for the rapid analysis of rhamnolipids in a biotechnological process with good reproducibility in sample determination and sufficient accuracy.An improvement in accuracy through continuous expansion and validation of the reference spectra set seems very likely.

Affiliation:
Research University Karlsruhe, Institute of Engineering in Life Sciences, Section of Technical Biology, Engler-Bunte-Ring 1, 76131 Karlsruhe, Germany. Rudolf.Hausmann@tebi.uni-karlsruhe.de.

ABSTRACT

Background: Vibrational spectroscopic techniques are becoming increasingly important and popular because they have the potential to provide rapid and convenient solutions to routine analytical problems. Using these techniques, a variety of substances can be characterized, identified and also quantified rapidly.

Results: The rapid ATR-FTIR (Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy) in time technique has been applied, which is suitable to quantify the concentrations of microbial rhamnolipids in a typical cultivation process. While the usually applied HPLC analysis requires an extensive and time consuming multi step extraction protocol for sample preparation, the ATR-FTIR-method allows the quantification of the rhamnolipids within 20 minutes. Accuracies between 0.5 g/l - 2.1 g/l for the different analytes were determined by cross validation of the calibration set. Even better accuracies between 0.28 g/l - 0.59 g/l were found for independent test samples of an arbitrarily selected cultivation.

Conclusion: ATR-FTIR was found to be suitable for the rapid analysis of rhamnolipids in a biotechnological process with good reproducibility in sample determination and sufficient accuracy. An improvement in accuracy through continuous expansion and validation of the reference spectra set seems very likely.